Dopaminergic neurons are involved in a variety of normal brain functions; degenerations of these neurons cause diseases in human. Investigation of how dopaminergic neurons respond to extracellular signals and molecular mechanisms regulating dopaminergic neuron survival and death often requires reliable, reproducible, and high-quality primary cultures of dopaminergic neurons. Here, we described methods to dissect and culture these neurons from embryonic mesencephalon of mouse brain. We utilize coverslips made from Aclar film to maximize the number of surviving dopaminergic neuron in the culture and immunocytochemistry of tyrosine hydroxylase (TH) to identify dopaminergic neuron.